At the cellular level, the principal defect in cystic fibrosis (CF) appears to be the abnormal regulation of Cl- transport across the apical membrane of airway epithelia. Apical CI- channels on the epithelia of CF patients are insensitive to stimulation by the Ca 2, or cAMP- dependent pathways which activate Cl- conductances in normal cells. Although the most common genetic defect in CF occurs within the cystic fibrosis transmembrane regulator (CFTR), the relationship between this protein and the regulation of apical Cl- conductances is unknown. The proposed research will employ techniques of molecular genetics and electrophysiology to isolate molecular clones encoding Cl- channels and study the regulatory interactions between Cl- channels and the cAMP and Ca2+ second messenger pathways. These studies will provide important information regarding the structure of Cl- channels and the mechanisms by which these channels are regulated in normal and CF afflicted cells. The primary structures of CI- channels remain unknown. To obtain molecular clones encoding CI- channels, size-fractionated mRNA, isolated from a cell line expressing these Cl- channels, has been identified which directs the expression of Cl- conductances following injection and two electrode voltage clamp of Xenopus oocytes. A cDNA expression library derived from this mRNA will be transcribed in vitro and similarly analyzed in oocytes: this will allow the identification of clones encoding Cl- channels.These studies will provide the first information regarding the primary structure and potential regulatory domains of Cl- channels. The molecular mechanisms by which Cl- channels are regulated are poorly understood. To determine the mechanisms of channel regulation, molecular clones encoding Cl- channels will be expressed alone or in the presence of cloned beta-adrenergic or muscarinic acetylcholine receptors The effect of beta-adrenergic stimulation (coupled to cAMP increase), muscarinic stimulation (coupled to Ca2+ increase) and membrane potential on Cl- channels will be analyzed by patch clamp of transfected mammalian cells or voltage clamp of Xenopus oocytes. In tandem, CF-affected skin fibroblast cells will be studied under patch clamp to assess the physiologic relevance of expression studies. Site-directed mutagenesis of the cloned Cl- channel will ultimately allow identification of the critical structures involved in channel regulation.